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United States Department of Agriculture

Agricultural Research Service

Research Project: Biorefining Processes

Location: Bioproducts Research

2011 Annual Report

1a. Objectives (from AD-416)
Objective 1: Develop enzyme-based technologies (based on cleaving specific covalent crosslinks which underlie plant cell wall recalcitrance) thereby enabling new commercially-viable* saccharification processes. Objective 2: Develop new enzyme-based technologies that enable the production of commercially-viable* coproducts such as specialty chemicals, polymer precursors, and nutritional additives/supplements from raw or pretreated lignocellulosic biomass. Objective 3: Develop pretreatment technologies that enable commercially-viable* biorefineries capable of utilizing diverse feedstocks such as rice straw, wheat straw, commingled wastes (including MSW), sorghum, switchgrass, algae, and food processing by-products. Objective 4: Develop new separation technologies that enable commercially-viable* and energy-efficient processes for the recovery of biofuels, biorefinery co-products, and/or bioproducts from dilute fermentation broths.

1b. Approach (from AD-416)
Novel enzymes for pretreatment of lignocellulosic feedstocks will be developed and improved by (1) creation of new genomic DNA libraries from diverse environments that are known to contain microbes that digest plant biomass, (2) development of novel rapid screening assays for identifying enzymes that have a specific activity, and (3) optimization of different enzyme cocktails for different biomass sources via multivariant, combinatorial optimization protocols. Greener routes toward production of styrene, terephthalic acid, vanillin and ferulic acid derivatives will be developed by a combination of biochemical and chemical synthetic pathways. Enzymes will be applied to create these bioproduct feedstocks. Engineering process models, economic analysis, and process parameters for developing integrated biorefineries using biomass from MSW and other under-utilized biomass sources as feedstock will be developed to create a source of cellulose that is consistent, easily converted to bioenergy and available during all seasons. Develop novel separation methods to reduce energy use and costs for recovering and purifying biofuels/bioproducts from low concentration fermentation broths, especially those resulting from lignocellulosic feedstocks where product concentrations are typically below (sometimes far below) 6 wt%. Replacing 5325-41000-046-00D (11/09).

3. Progress Report
Xylosidase activity has recently been recognized as being a critical rate-limiting component of commercial cellulosic biomass saccharification enzyme cocktails, and as such these commercial cocktails are now in many cases spiked with additional xylosidase activity. We discovered that xylosidase XylBH43, developed in Albany, has the second largest kcat reported for xylobiose hydrolyis, implying its high commercial potential. Conceived and developed an E. coli in-vivo whole-cell active/inactive (digital) 1st-tier high-throughput screening assay utilizing fluorophore-tagged substrates, for (1) gene discovery and (2) random-mutagenesis derived library purification for enzyme engineering applications. Demonstrated its use for the following enzyme classes that are critical for enzymatic hydrolysis of biomass: (1) xylosidases; (2) arabinofuranosidases, which remove arabinose residues from xylan chains; (3) xylanases, which hydrolyze the hemicellulose backbone; (4) cellulases, which hydrolyze the internal beta-1,4-bonds of cellulose; and (5) ferulic acid esterases, which hydrolyze ferulic acid groups that link lignin with the the hemicellulose backbone. A novel fabrication method was developed to make thin-film membranes for recovering alcohols from fermentation broths. The membranes show better performance than commercial membranes. A patent application was filed. CRADA to study mixed matrix membranes for recovery of ethanol from fermentation broths was completed and closed. A terminal report was issued. CRADA partner has initiated manufacturing of its first commercial product, which is used to dry water-soaked books and documents. The product contains a superabsorbent originally invented by USDA. New classes of adsorbents for use in controlling humidity in closed environments were studied. A collaboration with Reliant Energy was started, with the aim of developing a process to ferment almond hulls and other agricultural wastes to ethanol. Studies were initiated to characterize and analyze feedstocks.

4. Accomplishments
1. Improved ethanol separation from biomass fermentation using polymer membranes. In production of fuel ethanol from agriculturally-derived biomass, the recovery of ethanol from fermentation broths via distillation is energy-intensive with separation costs representing a high percentage of total cost. ARS researchers in Albany, CA, have invented a new process to make alcohol-selective membranes with performance significantly better than that of commercial membranes. Application of these new membranes reduces energy costs from cellulose-ethanol fermentation, especially for broths with low ethanol concentration. The improved performance of the alcohol-selective membranes makes them more cost-competitive for fuel ethanol operations, an advantage that makes them suitable for commercial application in the cellulose-ethanol industry.

2. Biorefineries that work at township- or farm-scale to convert agriculturally-derived biomass to bioenergy. Recent investment into biomass-to-ethanol biorefineries have been hampered by the large capital investment required to build very large facilities. ARS researchers in Albany, CA, have developed flexible technology that converts ag-derived biomass into bioenergy and chemical feedstocks by applying anaerobic digestion in concert with ethanol fermentation depending on feedstock type and scale. Specifically, improvements in anaerobic digestion to produce biogas and/or commercial acids, have resulted in process flexibility where the biorefinery can be sustainable at a small or large scale depending on feedstock availability. This flexibility allows scaling of the biorefinery for optimization at farm- or township-scale operation, with revenue generated by anaerobic digestion complementing cellulose-to-ethanol production.

3. Improved enzyme “cocktails” for breaking down ag-derived biomass. Industrial enzymes to break down agriculturally-derived biomass have proven costly because of the presence of complex, hard-to-degrade chemical crosslinks within straw and woody materials. ARS scientists in Albany and Peoria, along with researchers at U. Kentucky have combined efforts to improve an enzyme, xylosidase, that specifically breaks-down one of these complex “minor” linkages that are so critical in degrading biomass. This work not only resulted in one of the most active xylosidase enzymes ever reported in the literature but also development of high-throughput screening assays for this enzyme class, emonstration that this enzyme can be selectivity improved for reduced product inhibition, and clear evidence that these class of enzymes can work synergistically with other similar enzymes to degrade cellulose-rich biomass. This information is important for the cellulose-ethanol industry and can be used by enzyme companies to improve their enzyme biomass-degrading “cocktails."

Review Publications
Offeman, R.D., Ludvik, C.N. 2011. Poisoning of mixed matrix membranes by fermentation components in pervaporation of ethanol. Journal Membrane Science. 367:288-295.

Jha, A.K., Chen, L., Offeman, R.D., Balsara, N.P. 2011. Effect of nanoscale morphology on selective ethanol transport through block copolymer membranes. Journal Membrane Science. 373:112-120.

Robertson, G.H., Offeman, R.D., Cao, T., Orts, W.J. 2011. Ethanol in biorefining and dehydration of agricultural materials: energy, capital cost, and product quality implications. Biofuels, Bioproducts, & Biorefining (Biofpr). 5:37-53.

Sasagawa, T., Moriya, S., Lee, C.C., Kitamoto, K., Arioka, M. 2011. A high-throughput protein expression system in Pichia pastoris using a newly developed episomal vector. Plasmid Journal. 65(1):65-69.

Wong, D., Chan, V.J., Batt Throne, S.B., Sarath, G., Liao, H. 2011. Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass. Journal of Industrial Microbiology and Biotechnology. Epub ahead of print. DOI: 10.1007/s10295-011-0985-9.

Wagschal, K.C., Jordan, D.B., Braker, J.D. 2011. Expression, characterization, and site-directed mutagenesis of ß-D-xylosidase XylBH43 from Bacillus halodurans C-125. Process Biochemistry. doi:10.1016/j.procbio.2011.07.009.

Wong, D., Batt Throne, S.B., Robertson, G.H., Lee, C.C., Wagschal, K.C. 2010. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion. Industrial Biotechnology. 6:112-118.

Orts, W.J., Holtman, K.M., Seiber, J.N. 2008. Agricultural chemistry and bioenergy. Journal of Agricultural and Food Chemistry. 56:3892-3899.

Zhanmin, F., Wagschal, K.C., Wei, C., Montross, M., Lee, C.C., Yuan, L. 2009. Multimeric hemicellulases facilitate biomass conversion. Proceedings of the National Academy of Sciences. 75(6):1754-1757.

Wong, D. 2010. Applications of metagenomics for industrial bioproducts. Wong, D. Applications of metagenomics for industrial bioproducts. In: Marco, D., editor. Metagenomics: Theory, methods and applications. Norfolk, UK. Caister Academic Press.p. 141-158.

Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies. Applied Microbiology and Biotechnology. 86:1463-1471.

Wong, D., Chan, V.J., Mccormack, A.A., Batt Throne, S.B. 2010. Cloning and characterization of an exo-xylglucanase from rumenal microbial metagenome. Protein and Peptide Letters. 17:803-808.

Lee, C.C. 2010. Screening assays for biomass-degrading enzymes. Biofuels. 1(4):575-588.

Wong, D., Chan, V.J., Mccormack, A.A. 2009. Functional cloning of an endo-alpha-1,5-L-arabinanase gene from a metagenomic library. Protein and Peptide Letters. 16:1411-1435.

Lee, C.C., Kibblewhite, R.E., Wagschal, K.C., Robertson, G.H., Orts, W.J. 2009. Cloning and characterization of an alpha-glucuronidase from a mixed microbial population. Enzyme and Microbial Technology. 155(1-3):314-320.

Li, R., Zhang, Y., Lee, C.C., Liu, L., Huang, Y. 2011. HILIC separation mechanisms of tetracyclines on amino bonded silica column. Journal of Separation Science. 34(13):1508-1516.

Last Modified: 06/21/2017
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